The determination of the activity of creatine kinase (abbreviated herein to CK, but also known as creatine phosphokinase, CPK, or ATP:creatine phosphotransferase E.C.2.7.3.2.) in human serum is considered one of the most sensitive laboratory methods for diagnosing diseases of skeletal muscles, such as diseases of the myocardium. Total CK determination is useful, for example, for diagnosis of progressive muscular dystrophy, dermatomyositis and especially myocardial infarctions.
Conventional assays for total creatine kinase (i.e. assay for all isoenzymes) generally use either forward or reverse reaction illustrated by the following equation: EQU creatine+adenosine-5'-triphosphate (ATP).revreaction.creatine phosphate+adenosine-5'-diphosphate (ADP).
Both the forward and reverse reactions have been used in analytical procedures, but use of the reverse reaction is preferred because it is about six times faster than the forward reaction. The presence of ATP can then be determined by a number of colorimetric or potentiometric methods.
CK occurs in human body fluids and tissue in the form of three isoenzymes: CK-MM, for example in muscles, CK-BB, for example in the brain, and CK-MB, for example in the myocardium. The CK activity occurring in healthy human blood serum is normally due to the CK-MM isoenzyme, because CK-BB does not generally pass into the blood stream. In a healthy individual, CK-MB is generally restricted to certain organs, e.g. the myocardium. However, when the myocardium is damaged, as in the case of a cardiac infarction, CK-MB is released into the blood serum and can be detected therein.
A potential difficulty encountered in methods for determining CK-MB in biological fluids is interference from the other two isoenzymes. For practical pusposes, the amount of CK-BB in the fluid is considered negligible in most determinations. In methods for determining CK-MB, it is known to precipitate or inhibit the M subunit with specific antibodies to eliminate the interference of CK-MM and then to measure the remaining hybrid isoenzyme CK-MB.
A relatively recent contribution to clinical chemistry was the development of dry multilayer analytical elements useful for the assay of liquids. Such elements are described, for example, in U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al). These elements generally have a support on which is coated a registration layer and a porous spreading layer. The registration layer generally comprises a polymeric binder (e.g. gelatin) and optionally, one or more other components, including reagents, buffers, surfactants, etc.
A number of multilayer analytical elements have been designed for various assays and used commercially, including an element useful for the determination of total CK. Such elements have generally been prepared with about 5.5 g/m.sup.2 or less (dry coverage) of binder material in the registration layer in order to make as thin an element as possible and to reduce manufacturing costs.
It has been found, however, that such elements have reduced stability and room temperature keeping properties in high humidity environments, i.e. 50% relative humidity at 25.degree. C. Moreover, manufacturing processes used in making such analytical elements are necessarily carried out in this type of environment. The reduced stability under such conditions is a serious problem. It limits the flexibility in handling the elements by both the manufacturer and the user. The manufacturer must try to limit the amount of time the element is subject to high humidity. Further, if a user accidentally leaves the element out of the freezer compartment it is normally kept in prior to use, the element is likely to give erroneous results in the assay.
It would be desirable to improve the stability, and hence the room temperature keeping properties, of analytical elements in high humidity environments.